Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis

Long-standing models propose that plant growth responses to light or gravity are mediated by asymmetric distribution of the phytohormone auxin. Physiological studies implicated a specific transport system that relocates auxin laterally, thereby effecting differential growth; however, neither the molecular components of this system nor the cellular mechanism of auxin redistribution on light or gravity perception have been identified. Here, we show that auxin accumulates asymmetrically during differential growth in an efflux-dependent manner. Mutations in the Arabidopsis gene PIN3, a regulator of auxin efflux, alter differential growth. PIN3 is expressed in gravity-sensing tissues, with PIN3 protein accumulating predominantly at the lateral cell surface. PIN3 localizes to the plasma membrane and to vesicles that cycle in an actin-dependent manner. In the root columella, PIN3 is positioned symmetrically at the plasma membrane but rapidly relocalizes laterally on gravity stimulation. Our data indicate that PIN3 is a component of the lateral auxin transport system regulating tropic growth. In addition, actin-dependent relocalization of PIN3 in response to gravity provides a mechanism for redirecting auxin flux to trigger asymmetric growth.     

AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis

Polarized cellular distribution of the phytohormone auxin and its carriers is essential for normal plant growth and development. Polar auxin transport is maintained by a network of auxin influx (AUX) and efflux (PIN) carriers. Both auxin transport and PIN protein cycling between the plasma membrane and endosomes require the activity of the endosomal GNOM; however, intracellular routes taken by these carriers remain largely unknown. Here we show that Arabidopsis thaliana SORTING NEXIN 1 (AtSNX1) is involved in the auxin pathway and that PIN2, but not PIN1 or AUX1, is transported through AtSNX1-containing endosomes. We demonstrate that the snx1-null mutant exhibits multiple auxin-related defects and that loss of function of AtSNX1 severely enhances the phenotype of a weak gnom mutant. In root cells, we further show that AtSNX1 localizes to an endosomal compartment distinct from GNOM-containing endosomes, and that PIN2 accumulates in this compartment after treatment with the phosphatidylinositol-3-OH kinase inhibitor wortmannin or after a gravity stimulus. Our data reveal the existence of a novel endosomal compartment involved in PIN2 endocytic sorting and plant development.     

拟南芥PIN2介导的生长素极性运输调控植物根向地性

主要观察了拟南芥生长素输出载体PIN2及其介导的极性运输、生长素诱导合成对根尖生长素不对称分布和根向地性反应的影响.结果表明:拟南芥PIN2基因突变、诱导内源生长素IAA及用抑制剂NPA或TIBA抑制生长素极性运输都严重影响了根尖生长素不对称分布的形成,最终抑制植物根向地性反应,暗示PIN2介导的生长素极性运输和生长素不对称分布在根向地性反应中起着关键性调控作用.从这些研究结果可进一步理解生长素调控植物根向地性的分子机理.     

Auxin transport inhibitors block PIN1 cycling and vesicle trafficking

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.     

生长素调控极性运输载体PIN2质膜丰度与降解

主要观察了外源生长素长时间(8 h)处理对拟南芥生长素极性输出载体PIN2-GFP质膜丰度的影响,低温、KCN处理和生长素受体突变对生长素调控质膜PIN2-GFP丰度的影响,以及液泡H+-ATPase抑制剂ConA对野生型和生长素受体四突变体tir1afb1,2,3液泡中PIN2-GFP积累的影响.结果表明:外源生长素通过其受体TIR1/AFB介导的信号途径下调质膜PIN2-GFP的丰度,促进PIN2-GFP的内吞、胞内运输和液泡降解.暗示生长素通过TIR1/AFB介导的信号途径反馈调控质膜PIN2-GFP的水平,从而阻止了胞内生长素的过多输出.     

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.     

Auxin inhibits endocytosis and promotes its own efflux from cells

One of the mechanisms by which signalling molecules regulate cellular behaviour is modulating subcellular protein translocation. This mode of regulation is often based on specialized vesicle trafficking, termed constitutive cycling, which consists of repeated internalization and recycling of proteins to and from the plasma membrane. No such mechanism of hormone action has been shown in plants although several proteins, including the PIN auxin efflux facilitators, exhibit constitutive cycling. Here we show that a major regulator of plant development, auxin, inhibits endocytosis. This effect is specific to biologically active auxins and requires activity of the Calossin-like protein BIG. By inhibiting the internalization step of PIN constitutive cycling, auxin increases levels of PINs at the plasma membrane. Concomitantly, auxin promotes its own efflux from cells by a vesicle-trafficking-dependent mechanism. Furthermore, asymmetric auxin translocation during gravitropism is correlated with decreased PIN internalization. Our data imply a previously undescribed mode of plant hormone action: by modulating PIN protein trafficking, auxin regulates PIN abundance and activity at the cell surface, providing a mechanism for the feedback regulation of auxin transport.     

脱落酸对水稻根系生长素合成与运输的调控

利用一系列不同浓度的脱落酸(ABA)处理水稻幼苗根系,观察水稻初生根根长、冠根数目、侧根数目及长度、DR5::GUS转基因材料GUS活性、生长素的合成及运输相关基因表达水平等变化.结果表明,ABA可以抑制水稻初生根的伸长、冠根和侧根的发生和伸长,并且抑制效应呈现剂量效应.DR5::GUS活性在整个根系中随ABA处理浓度升高而减弱,但根尖部位的DR5::GUS活性经过ABA处理后表现增强;生长素合成相关基因YUCCA2、YUCCA4、YUCCA7,生长素运输载体基因AUX3、AUX5、PiN1b、PiN2、PiN5a、PiN9、PiN10a均显著下降表达.     

An <Emphasis Type="Italic">Arabidopsis ctpA</Emphasis> homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease （CtpA） homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

过氧化氢对拟南芥生长素信号传导相关蛋白的影响

本论文通过添加外源过氧化氢来探究其对植物生长素信号的影响。以GUS基因作为报告基因,研究拟南芥中各生长素相关蛋白(生长素含量标志蛋白DR5、输入蛋白AUX1、输出蛋白PIN1和PIN2)对过氧化氢的响应。结果表明过氧化氢处理后拟南芥呈植株变小、根变短和不定根数目增多的表型;与对照相比,GUS染色后各生长素相关蛋白的表达下降,这表明过氧化氢对植物生长素的合成与运输起一定的抑制作用。在过氧化氢处理的基础上,添加抗氧化物质发现植株表型有明显的恢复,GUS染色也表明相关蛋白的表达量均增加;另外,0.0001 ?mol/L的外源IAA也能恢复过氧化氢处理后拟南芥的表型和生长素相关蛋白的表达。综上所述,过氧化氢主要通过抑制植物体内的生长素信号来调控植物的生长发育。     

Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Proteomic analysis of long-term salinity stress-responsive proteins in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> leaves

Salinity is one of the most severe environmental factors that may impair crop productivity. A proteomic study based on two-dimensional gel electrophoresis is performed in order to analyze the long-term salinity stress response of Thellungiella halophila, an Arabidopsis-related halophyte. Four-week-old seedlings are exposed to long-term salinity treatment. The total crude proteins are extracted from leaf blades, separated by 2-DE, stained with Coomassie Brilliant Blue, and differentially displayed spots are identified by MALDI-TOF MS or QTOF MS/MS. Among 900 protein spots reproducibly detected on each gel, 30 spots exhibit significant change and some of them are identified. The identified proteins include not only some previously characterized stress-responsive proteins such as TIR-NBS-LRR class disease resistance protein, ferritin-1, and pathogenesis-related protein 5, but also some proteins related to energy pathway, metabolism, RNA processing and protein degradation, as well as proteins with unknown functions. The possible functions of these proteins in salinity tolerance of T. halophila are discussed and it is suggested that the long-term salinity tolerance of T. halophila is achieved, at least partly, by enhancing defense system, adjusting energy and metabolic pathway and maintaining RNA structure.     

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Temperature-sensitive gold-nanotube array membranes modified with poly（N-isopropylacrylamide）

A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly（N-isopropylacrylamide） （PNIPAm）. The permeation ability of the mem-branes at different temperatures was investigated using sodium fluorescein and quantum dots as probes. The results showed that the pore diameter of nanotube was changed due to the reversible response of PNIPAm-modified membranes to temperature, and then the permeation ability of the mem-branes was changed. The permeation of fluorescence probes was slow and even almost blocked at 25℃ （below the lower critical solution temperature, LCST）, since PNIPAm formed expanded structures and decreased the pore size. While at 40℃ （above the LCST）, the permeation was increased, since PNIPAm became compact structures and the pore diameter was increased. Furthermore, the permeation ability of the temperature-sensitive nanotube array membranes could be adjusted reversibly and it is possible to use the membranes in nanofluidic devices, nanogates, etc.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

Transport, capture and exocytosis of single synaptic vesicles at active zones

To sustain high rates of transmitter release, synaptic terminals must rapidly re-supply vesicles to release sites and prime them for exocytosis. Here we describe imaging of single synaptic vesicles near the plasma membrane of live ribbon synaptic terminals. Vesicles were captured at small, discrete active zones near the terminal surface. An electric stimulus caused them to undergo rapid exocytosis, seen as the release of a fluorescent lipid from the vesicles into the plasma membrane. Next, vesicles held in reserve about 20 nm from the plasma membrane advanced to exocytic sites, and became release-ready 250 ms later. Apparently a specific structure holds vesicles at an active zone to bring v-SNAREs and t-SNAREs, the proteins that mediate vesicle fusion, within striking distance of each other, and then allows the triggered movement of such vesicles to the plasma membrane.